Serious infection with Staphylococcus aureus (SA) remains an important clinical challenge despite potent antibiotics. Novel therapeutic advances await elucidation of the molecular bases for persistence, chronicity, and metastatic spread - i.e. the hallmarks of SA infection. Overwhelming infection with virulent strains and increasing antibiotic resistance are powerful incentives to understand better the host defense against SA. Polymorphonuclear neutrophils (PMN) represent the cornerstone of cell-mediated antimicrobial activity and exert ~ all of their antimicrobial effort within phagosomes, where reactive oxygen species (ROS) and granule contents collaborate to kill and degrade microbes. Importantly, hydrogen peroxide (H2O2) produced by PMN is amplified by the PMN granule protein myeloperoxidase (MPO) to generate HOCl (bleach). In addition to PMN, a specific Group IIA phospholipase A2 (GpIIA-PLA2), which is present in plasma of infected animals, tears, and inflammatory fluid, exhibits potent activity to kill and degrade SA. With VA Merit support, we have made progress in elucidating features of two complementary aspects of interactions between ingested SA and PMN, demonstrating (a) a synergy between PMN-dependent ROS and GpIIA-PLA2 to kill and degrade SA, and (b) several characteristics of MPO-H2O2-Cl attack on SA in phagosomes. Furthermore, we have identified transcriptional and structural responses by SA immediately following phagocytosis. We suspect that such changes contribute to the capacity of some ingested SA to survive in PMN and subsequently escape, phenomena we have examined and are consistent with longstanding clinical observations and experimental data . We propose now to use tools that we have created and analytical methods we have developed during the previous period of VA funding to extend our novel studies and test the overall hypothesis that the responses of SA in the PMN phagosome to modify the composition of their cell surface (including content of D-alanine and cardiolipin) and to induce cytoplasmic anti-oxidants (e.g. methionine sulfoxide reductase and hsp33) result in their capacity to resist actions of PMN- GpIIA-PLA2 and the specific toxicity of HOCl and related oxidants, and to survive in, and escape from, PMN and perpetuate infection. Our Specific Aims are: 1. To define the specific contributions of the PMN oxidase-derived oxidants to the synergy of human PMN and GpIIA-PLA2 against SA What MPO-mediated modifications of SA proteins and phospholipids occur during phagocytosis? What modifications in SA phospholipids and proteins induced by ROS 1 MPO in the phagosome alter GpIIA- PLA2, its substrates, or both? Are genetic mutants in cell wall constituents, including D-alanylation or cardiolipin synthase, better equipped to survive in and escape from the PMN phagosome? 2. To determine how the MPO-H2O2-Cl system kills most SA and, conversely, how the subset of surviving organisms adapt to respond to overcome MPO-derived cytotoxins in the phagosome. Does bleaching of cytoplasmic GFP in SA provide accurate assessment of HOCl activity in PMN phagosome? What proteins in SA are targets for MPO-specific modifications;which contribute, directly or indirectly, to susceptibility of SA to PMN? What targets are repaired by phagocytosed SA? Are SA with mutations in methionine sulfoxide reductases and the redox-sensitive chaperone hsp33, systems that respond to HOCl-induced oxidant stress, more or less vulnerable to cytotoxins in PMN phagosomes? Does resistance to HOCl-mediated damage allow SA to persist in or escape from PMN? We anticipate that our studies will provide important and novel insights into the complex biology that occurs when ingested SA meet the cytotoxic contents of the PMN phagosome. In addition, we believe that novel targets for therapeutic intervention may be identified as a result of our proposed work. PUBLIC HEALTH RELEVANCE: SA infections occur commonly in Veterans, both in outpatients as well as hospitalized patients. The VA healthcare system served >5 million people in 2007 with ~580,000 discharges from 153 medical centers and is thus one of the largest healthcare systems in the US. According to a VA-based study, the prevalence of SA infections increased from 26.93 per 1,000 discharges in 2000 to 30.59 in 2007 ( 14%). Consequently, the attendant rises in morbidity and mortality present a significant challenge to our health care system. Understanding the mechanisms by which innate immune elements, including PMN and GpIIA-PLA2, mediate successful killing and degradation of SA will provide fundamental insights that can be translated into the development of novel targets for therapy. We believe that our eludication of responses that allow SA to survive in PMN, and in some cases escape, may reveal genes and their products that contribute to virulence but are not required for viability per se, thus eluding more conventional assays to identify antibiotic targets.